The in-vivo imaging of tumor vasculature in rodents can provide invaluable information about tumor development and treatment efficacy. Imaging in vivo fine micro-vascular structure and function presents a challenge to existing small animal imaging techniques including micro-CT and micro-MRI due to combined requirements for high spatial and temporal resolution. We propose the use of Tomographic Digital Subtraction Angiography (TDSA) for estimation of tumor perfusion in small animals. TDSA combines the advantages of high temporal resolution of digital subtraction angiography (DSA) and high spatial resolution of micro-CT. TDSA augments projection imaging methods such as DSA by providing three-dimensional information using tomosynthesis and CT reconstruction algorithms. TDSA approach is based on the novel paradigm that the same time density curves can be reproduced in a number of consecutive injections of microL volumes of contrast at a series of different angles of rotation. We will build a TDSA system with two tubes/detectors to provide data for 4D imaging with a temporal resolution on the order of 100 ms and spatial resolution ranging from 100 microns to 1 mm. This represents a ten fold increase in temporal resolution and at least two times higher resolution than those achieved by the state of the art pre-clinical micro-CT systems used in perfusion imaging in small animals. The capabilities of TDSA will be established in studies on tumor physiology in both rats and mice. TDSA perfusion measures will be calibrated and validated using a dynamic phantom and techniques exploiting microspheres and vascular staining. We will integrate TDSA with conventional micro-CT to provide a standardized protocol for characterization of tumor perfusion, permeability and 3D vascular architecture, using a combination of conventional and blood pool iodinated contrast agents. TDSA will provide the 4D functional imaging method capable of achieving the very high temporal and spatial resolution required for studies on tumor physiology and evaluation of anti-angiogenic drugs on small animals. [unreadable] [unreadable] [unreadable]